Methylamine metabolism in Hansenula polymorpha: an in vivo 13C and 31P nuclear magnetic resonance study

Abstract

Methylamine uptake, oxidation, and assimilation were studied in Hansenula polymorpha, a methylotrophic yeast. The constitutive ammonia transport system was shown to be effective at accumulating methylamine within cells cultured with methylamine or ammonia as a nitrogen source. [13C]methylamine oxidation rates were measured in vivo in methylamine-adapted cells by 13C nuclear magnetic resonance and were found to be lower than its uptake rate into the cells. The 13C label of methylamine was found exclusively in trehalose and glycerol, and [13C]formaldehyde was also extensively assimilated, indicating the presence of an assimilation pathway for the methylamine carbon. In vivo 31P nuclear magnetic resonance analysis showed major differences in the endogenous polyphosphate levels and mean chain length during adaptation of the cells from ammonia to methylamine, indicating that methylamine accumulated in the vacuole in the same manner as basic amino acids and purines. [13C]glucose metabolism was drastically altered during adaptation of the cells from ammonia to methylamine as a nitrogen source. The total rate of glucose utilization and the rate of ethanol production fell. Direct trehalose synthesis from glucose increased, indicating a switch from carbon utilization for growth to that for storage. The rate of methylamine oxidation was sufficient to support a much higher flow of carbon into central biosynthetic pathways. These results suggest that this reduction in biosynthetic carbon flow, rather than nitrogen availability, was the main factor responsible for reducing the growth rate of the yeast when ammonia was replaced by methylamine as the nitrogen source.